Analysis of leukocyte subpopulations from whole blood samples is an integral and essential part of diagnostic procedures regarding a multiplicity of pathologies. The ability to analyze the major subpopulations of leukocytes in an automated manner is essential for a rapid diagnosis of a single blood sample and for the rapid processing of many samples at once. In addition, the determination of hemoglobin concentration provides useful diagnostic information about the health of a patient.
Traditional diagnosis of blood samples involves the smearing of a blood sample on a microscope slide, followed by manual visual analysis of the individual slide. This approach is extremely time-consuming as well as being subjective to the interpretation of the individual analyzing the slide. These factors have led to the development of automated leukocyte analysis utilizing flow cytometry. In automated leukocyte analysis using hematology instruments, the red blood cells are lysed which enables the differentiation of the leukocytes and determination of hemoglobin concentration.
U.S. Pat. No. 4,286,963 (to Ledis et al.) describes a method for achieving rapid hemolysis of red blood cells in whole blood and automated analysis of lymphoid and myeloid subpopulations of leukocytes, and quantitative determination of hemoglobin using potassium cyanide.
U.S. Pat. No. 4,485,175 (to Ledis et al.) describes a method for performing differential determinations of leukocytes into three (3) subpopulations utilizing automated cell counting equipment. However, this method is limited to effect differentiation of the leukocytes into three subpopulations: lymphocytes, monocytes and granulocytes.
These above-mentioned methods not only lyse red blood cells, but also destroy leukocyte membranes. The differentiation, consequently, is based on the nuclear volumes of the leukocyte subpopulations. The application of these methods, alone or in combination with other means, prohibits further refinement in the diagnostic process of various disease states based on the differences in the immunochemical response of the surface marker of the cell membrane.
U.S. Pat. No. 5,155,044 (to Ledis et al.) discloses a method for isolation and analysis of leukocytes from a whole blood sample, which enables automated differentiation of leukocytes into five (5) subpopulations utilizing an automated hematology analyzer. However, this method cannot convert oxyhemoglobin of the sample to a stable chromogen for hemoglobin measurement. The oxyhemoglobin method is an unreliable method because of incomplete hemoglobin conversion.
U.S. Pat. No. 5,389,549 (to Hamaguchi et al.) describes a lysis reagent system which contains a nonionic polyoxyethylene surfactant. Using the method and lysis reagent system disclosed, it is difficult to do a full analysis of the five major leukocyte subpopulations. Full analysis of leukocyte subpopulations requires differential lysis of the red blood cells and leukocytes, and three separate determinations for the identity of eosinophil, neutrophil and basophil populations in addition to the lymphocyte and monocyte populations. Additionally, this system requires a hypotonic lysing environment which is extremely shocking to the cells and makes preservation of the cells in a near native state difficult.
In addition, measuring hemoglobin concentration in a blood sample is another diagnostic tool when doing blood analysis. Historically, hemoglobin determinations have been performed by forming and measuring cyanide hemoglobin. However, the reagent waste from this method is of enormous environmental concern. Several cyanide-free methods for lysing red blood cells and measuring hemoglobin have been developed. U.S. Pat. No. 5,250,437 (to Toda et al.) and U.S. Pat. No. 5,242,832 (to Sakata) all utilize quaternary ammonium salt lysis systems for hemolyzing red blood cells and oxidizing the hemoglobin. However, because of the harshness of the quaternary ammonium ion based systems on leukocytes, these systems cannot be used for combined leukocyte subpopulation differentiation greater than three subpopulations and hemoglobin determination, particularly if near native state, leukocyte differentiation is desired.
EPO No. 0 325 710 (to Hamaguchi et al.) discloses a method for the hemolysis of red blood cells. However, with this method, one can only differentiate three subpopulations in addition to measuring the oxyhemoglobin.
U.S. Pat. No. 5,516,695 (to Kim et al.) discloses a method for rapid analysis of a whole blood sample allowing the determination of five subpopulations of white blood cells, nucleated red blood cells, and lymphocyte immunophenotyping on automated hematology instrumentation. The disclosed method lyses red blood cells and concurrently fixes white blood cells and preserves surface antigens on lymphocytes. The preservation of white cell surface marker is based on fixation by an aliphatic aldehyde, which is known as an environmentally unfriendly chemical. In addition, the method requires heating of the reagent to about 40.degree. C. for leukocyte differential of a whole blood sample in order to achieve the performance and the throughput requirement of an automated hematology analyzer. Without heating, the lysing time is substantially longer.
U.S. Pat. Nos. 5,030,554 and 5,437,985 (to Quintana et al.) disclose a method to prepare a whole blood sample for photo-optical analysis of lymphocyte subpopulations. The method comprises a 3-step sample preparation of: (1) lysing red cells with an acid lyse; (2) quenching the lysing reagent by an alkaline quench solution; (3) fixing white cells with a fixative solution. Indicator binding can be accomplished before, concurrent with, and after the lysing. This method also utilizes aldehyde fixation to preserve white cells. In addition, the light scatter and fluorescence analyses as disclosed differentiate only three leukocyte subpopulations, i.e., monocytes, lymphocytes and granulocytes.